PROJECT ABSTRACT Cellular organelles form dynamic intracellular networks by engaging in direct membrane contact. Membrane contact sites (MCSs), which link organelle membranes via protein interactions, are fundamental to the regulation of organelle structure, composition, and dynamics. As these features dictate organelle function, MCSs also control diverse biological processes like, metabolism, trafficking, organelle biogenesis, and apoptosis. However, organelle contacts remain largely unexplored during mammalian virus infection despite the knowledge that organelles play critical, and often conserved, roles in a broad range of virus infections. Moreover, many virus- induced changes to organelles, such as vesicular remodeling, mitochondrial fragmentation, and immune modulation, are directly related to MCS-controlled functions. I hypothesize that membrane contact sites are key regulatory hubs of virus replication and actively modulated during infection. I propose an integrative live-cell imaging and quantitative proteomics approach to examine MCS dynamics across subcellular space and infection time. This workflow includes targeted proteomics to determine temporal MCS protein abundances, confocal microscopy to examine organelle contact phenotypes, and molecular virology-based functional assays that delve into the mechanisms underlying the roles of membrane contact sites in virus replication. I have already established an experimental method to track alterations in MCS protein abundances with high sensitivity and precision during the progression of a viral infection. I further applied this method in infections with several human viruses, including the herpesviruses cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1), and the orthomyxovirus Influenza A. Indeed, I discovered that these viruses change the composition of MCSs across infection time, with temporal and organelle specificity related to each unique virus replication strategy. Among these viruses, the wide-spread human pathogen HCMV caused the most striking changes, triggering a nearly global upregulation and rewiring of MCSs. Given the known role of the endoplasmic reticulum (ER) as a master regulator of cellular organelles, ER-mediated contacts are poised to facilitate key steps in the virus replication cycle, and my preliminary functional analyses support this. In this study, I will investigate the function and regulation of two critical ER contacts in HCMV infection. First, I will establish the role of ER-peroxisome contacts in modulating peroxisome plasticity for virus assembly. I hypothesize that ER-peroxisome contact is increased during infection and modified by virus-host protein interactions to enhance lipid synthesis for formation of the viral envelope. Second, I will investigate ER-endosome contacts as regulators of the vesicular rearrangements required for virion assembly and egress, which I predict occurs in an endosome type-specific manner to control cholesterol distribution and endosome trafficking directionality. As MCSs are fundamental to organelle biology, this work will help expand the understanding of the mechanisms at the core of human virus replication.